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. Author manuscript; available in PMC: 2014 Jun 25.
Published in final edited form as: Curr Protoc Microbiol. 2013 Feb;0 9:Unit–9C.2. doi: 10.1002/9780471729259.mc09c02s28

Laboratory Maintenance of Methicillin-Resistant Staphylococcus aureus (MRSA)

Nicholas P Vitko 1, Anthony R Richardson 1,*
PMCID: PMC4070006  NIHMSID: NIHMS594321  PMID: 23408135

Abstract

Staphylococcus aureus is an important bacterial pathogen in the hospital and community settings, especially Staphylococcus aureus clones that exhibit methicillin-resistance (MRSA). Many strains of S. aureus are utilized in the laboratory, underscoring the genetic differences inherent in clinical isolates. S. aureus grows quickly at 37°C with aeration in rich media (e.g. BHI) and exhibits a preference for glycolytic carbon sources. Furthermore, S. aureus has a gold pigmentation, exhibits β-hemolysis, and is catalase and coagulase positive. The four basic laboratory protocols presented in this unit describe how to culture S. aureus on liquid and solid media, how to identify S. aureus strains as methicillin resistant, and how to generate a freezer stock of S. aureus for long-term storage.

Keywords: Staphylococcus aureus, HA-MRSA, CA-MRSA, growth, strain selection, CDM, freezer stock

INTRODUCTION

Staphylococcus aureus is a gram-positive coccus (~0.6μm in diameter) that colonizes the skin and/or nares of most humans. Given access to other tissues (via tissue disruption or an impaired immune system), S. aureus can cause a wide variety of serious clinical manifestations including: pneumonia, osteomyelitis, endocarditis, skin and soft tissue infections (SSTI's), and septicemia. This unit describes methods, reagents, and equipment commonly utilized for the growth, maintenance, and characterization of S. aureus in the laboratory. Basic Protocol 1 describes the culturing of S. aureus on agar plates, Basic Protocol 2 describes the Oxacillin-salt agar method for determining methicillin-resistance of S. aureus strains, Basic Protocol 3 describes the culturing of S. aureus in liquid media, and Basic Protocol 4 describes the preparation of a frozen stock of S. aureus from a liquid culture. Additional information regarding strain selection (Table 1), unique molecular characteristics of CA-MRSA isolates (Table 2), selective antibiotic concentrations (Table 3), the appearance of cultured S. aureus (Figures 1–3), and a recipe for chemically defined media (Table 4) is also provided.

Table 1.

Commonly Utilized Stophylococcus aureus Strains

Strain Classification PFGE Type Sequenced Origin
NCTC8325 (RN1) HA-MSSA N/A Yes Corneal ulcer isolate from sepsis patient (1960).
NCTC8325-4 (RN450) MSSA N/A Yes Laboratory strain, derived from NCTC8325 by UV mutagenesis.
SH1000 MSSA N/A Yes Laboratory strain, derived from NCTC8325-4 by complementation of rsbU
RN6390 MSSA N/A No Laboratory strain, derived from NCTC8325-4.
RN4220 MSSA N/A Yes Laboratory strain, derived from NTC8325-4 using chemical mutagenesis (MNNG).
Newman HA-MSSA N/A Yes Osteomyelitis Isolate, TB patient (pre-1952)
COL HA-MRSA N/A Yes Clinical isolate, Colindale, England (1961)
Mu50 HA-MRSA USA100 Yes Vancomycin resistant SSI isolate from male infant, Japan (1996)
N315 HA-MRSA USA100 Yes Pharyngeal smear isolate, Japan (1982)
JH1 and JH9 HA-MRSA USA100 Yes Sequential blood isolates, vancomycin resistant infective endocarditis in patient with underlying congenital heart disease, Baltimore, USA (2003)
UAMS-1 HA-MRSA USA200 No Osteomyelitis Isolate, McClellan Veterans Hospital, Little Rock, Arkansas, USA (1995)
MRSA252 HA-MRSA USA200 Yes 64-year-old female, fatal post-op septicemia, United Kingdom (1997)
LAC CA-MRSA USA300 No SSTI Isolate, County Jail, Los Angeles, USA (2002)
SF8300 CA-MRSA USA300 No Wound Isolate, San Francisco, USA (1996–2003)
FPR3757 CA-MRSA USA300 Yes Wrist abscess isolate, 36-year old HIV-positive white male, San Francisco, California USA
TCH1516 CA-MRSA USA300 Yes Clinical isolate, septic arthritis, 14-year-old black male, Texas Children's Hospital, Houston, Texas, USA (2002)
MW2 CA-MRSA USA400 Yes Blood isolate, fatal septicemia, 16-month-old American Indian female, North Dakota, USA (1998)
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Table 2.

Unique CA-MRSA Genes/Attributes

Characteristic Description Exceptions
ACME Cassette Arginine Catabolic Mobile Element carried by most USA300 CA-MRSA isolates(contains 33 genes, including speG and a fully functional arc operon) USA300 clones in S. America
speG Encodes a spermine acetyltransferase on the ACME island. Provides spermine resistance. See Above
lukSF-PV Encodes for Panton-Valentine leukocidin or PVL (pore forming toxin). ST72 in Asia
sasX Encodes for surface anchored virulence factor involved in aggregation and attachment to host cells. Non-ST239 isolates
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Table 3.

Antibiotic Stock Solutions for Use with S. aureus Cultures

Antibiotics Solvent Stock Concentration Working Concentration
Chloramphenicol EtOH 20mg/ml 20 μg/ml
Erythromycin EtOH 5 mg/ml 5 μg/ml
Spectinomycin H2O 100 mg/ml 100 μg/ml
Kanamycin H2O 50 mg/ml 50 μg/ml
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Figure 1.

Figure 1

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Staphylococcus aureus strains SF8300 (right) and RN4220 (left) grown on Brain Heart Infusion agar for 18 hr at 37°C.

Figure 3.

Figure 3

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Growth of Staphylococcus aureus (SF8300, left; COL, middle) and E.coli (DH10B, right), in Brain Heart Infusion Broth for 18 hr at 37°C with aeration.

Table 4.

Chemically Defined Media for Growth of S. aureus Cultures.

Solution(s) Item Amount (g) Volume (ml) Solvent Sterilize
Salt Solution K2HPO4 70 1000 H2O Autoclave
KH2PO4 20
(NH4)2SO4 10
MgSO4⬆7H2O 25.6
Amino Acids Phe 1 250 1M NH4OH Autoclave
Iso 7.50E-01 250 1M NH4OH Autoclave
Tyr 1.25 250 1 N HCl Autoclave
Cys★ 5.00E-01 250 1 N HCl Autoclave
Glu 2.5 250 1 N HCl Autoclave
Lys 2.50E-01 250 1 N HCl Autoclave
Met 1.75 250 1 N HCl Autoclave
His 7.50E-01 250 1 N HCl Autoclave
Trp 2.50E-01 250 1 N HCl Autoclave
Leu 2.25 250 1 N HCl Autoclave
Asp 2.25 250 1 N HCl Autoclave
Arg 1.75 250 1 N HCl Autoclave
Ser 7.50E-01 250 dH2O Autoclave
Ala 1.5 250 dH2O Autoclave
Thr 7.50E-01 250 dH2O Autoclave
Gly 1.25 250 dH2O Autoclave
Val 2 250 dH2O Autoclave
Pro 2.50E-01 250 dH2O Autoclave
Bases★★ Adenine 1.25E-01 250 dH2O Autoclave
Cytosine 1.25E-01 250 dH2O Autoclave
Guanine 1.25E-01 250 dH2O Autoclave
Thymine 5.00E-01 250 dH2O Autoclave
Uracil 1.25E-01 250 dH2O Autoclave
Vitamin Solution Thiamine 1.00E-01 100 H2O Autoclave
Niacin 1.20E-01
Biotin 5.00E-04
Ca Pantothenate 2.50E-02
Trace Elements FeCl3 8.00E-01 100 H2O Filter Sterilize
ZnCl 6.95E-03
MnCl ⬆ 4H20 9.90E-03
Boric Acid 6.00E-04
CoCl2 ⬆ 6H20 3.97E-02
CuCl2 ⬆ 2H20 2.56E-04
NiCl2 ⬆ 6H20 2.38E-03
Na2MoO4 ⬆ 2H20 3.58E-03
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★

Cystine not cysteine

★★

May need to heat or titrate using KOH to get into solution

CAUTION: Staphylococcus aureus is a Biosafety Level 2 (BSL-2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. No special precautions are necessary when working with methicillin-resistant Staphylococcus aureus (MRSA). See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.

Strategic Planning

Unlike other pathogenic bacteria studied in the laboratory, there are many commonly utilized strains of S. aureus (Table 1). This is a reflection of the fact that S. aureus strains exhibit variation in commonly studied in vitro phenotypes, different degrees of virulence in mouse models of infection, and were isolated from a wide variety of infection sites and individuals. As mentioned previously, S. aureus is commonly divided into the methicillin-resistant (MRSA) and -susceptible subgroups (MSSA). MRSA strains were first isolated in the 1960's (Jevons, 1961) and it was later determined their resistance was linked to the acquisition of a mobile genetic element (staphylococcal cassette chromosome) harboring the mecA gene, (SCCmec) (Ubukata et al., 1989). Since the identification of the original SCCmec cassette, research on this topic has revealed the existence of other SCCmec cassette types that confer additional antibiotic resistance genes to S. aureus isolates (for a description of the SCCmec types see, (International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC), 2009)).

The most commonly studied MSSA isolates include: NCTC8325 (RN1), NCTC8325-4 (RN450), SH1000, RN6390, RN4220, and Newman. NCTC8325 (RN1) was isolated in 1960 from a sepsis patient and was primarily utilized in the laboratory for the propagation of phage 47 and the study of S. aureus genetics (NOVICK and RICHMOND, 1965). To make isolation of phage 47 easier, NCTC8325 was cured of three endogenous prophages encoded in its genome via successive rounds of UV mutagenesis, yielding strain NCTC8325-4 (RN450) (Novick, 1967; Herbert et al., 2010). Eventually, it became clear that NCTC8325 exhibited reduced toxin production, pigmentation, and virulence in mice, as compared to other clinical S. aureus isolates. Genomic comparison of NCTC8325 to other MSSA strains confirmed that this strain had mutations in rsbU (activator of sigB) and tcaR (activator of spa) (Herbert et al., 2010), making it a poor model for the study of S. aureus genetic regulation and virulence (albeit it has been widely utilized for the study of these two topics). To reverse this loss of virulence, the rsbU mutation was repaired in strain NCTC8325-4, yielding strain SH1000 (Horsburgh et al., 2002). Likewise, RN6390 is a more virulent derivative of NCTC8325-4 (produces greater amounts of exoprotein and α-toxin), but it was generated via sequential phage transduction (Peng et al., 1988).

Like SH1000 and RN6390, RN4220 is also a derivative of NCTC8325-4. However, RN4220 was mutagenized via nitroguanoside to enrich for mutants that would efficiently accept plasmid DNA from E. coli (Kreiswirth et al., 1983; Herbert et al., 2010). As a result, RN4220 carries 121 SNP's (eleven shared by NCTC8325-4), 4-large scale deletions, an insertion in agrA, and a nonsense mutation in hsdR (the major restriction endonuclease in S. aureus) (Nair et al., 2011) when compared to S. aureus NCTC8325. The agrA mutation present in RN4220 has been shown to lead to delayed activation of the agr quorum sensing system, which in turn, affects virulence factor production. Thus, RN4220 is not recommended for the study of S. aureus virulence regulation or in vivo pathogenesis. Instead, RN4220 is used as a cloning intermediate when moving plasmid DNA from E. coli into S. aureus.

The final MSSA isolate widely utilized in the laboratory is S. aureus Newman. Newman is not a derivative of NCTC8325, but was instead isolated from a secondary osteomyelitis infection of a TB patient prior to 1952 (DUTHIE and LORENZ, 1952). Like NCTC8325, and all of its derivatives, Newman has unique features, including: the overproduction of toxins resulting from the constitutive expression of saeRS (Adhikari and NOVICK, 2008) as well as mutations in genes encoding for the virulence proteins FnbA and FnbB that limits their surface localization (Grundmeier et al., 2004). Furthermore, the S. aureus Newman genome contains four prophages (ΦNM1-4), three of which (ΦNM1, 2, and 4) are known to replicate during in vitro culture and animal infections (Bae et al., 2006). Despite these observations, Newman is widely utilized for virulence studies of S. aureus.

The most commonly studied MRSA isolates include: COL, Mu50, N315, JH1, JH9, UAMS-1, MRSA252, LAC, SF8300, FPR3757, TCH1516, and MW2 (Table 1). All MRSA isolates are commonly divided into hospital and community acquired strains (HA and CA-MRSA) based on the origin of the disease-causing isolate. Interestingly, this distinction has also come to reflect historical development of S. aureus pathogenesis and the emergence of a new MRSA-epidemic. HA-MRSA was first isolated in 1961 and quickly became the dominant hospital acquired infection following the widespread introduction of methicillin treatment in hospitals (Jevons, 1961). The major HA-MRSA strains studied in the laboratory are COL (Sabath et al., 1972; Dyke, 1969), Mu50 (intermediate-vancomycin resistance) (Hiramatsu et al., 1997; Kuroda et al., 2001), N315 (Kuroda et al., 2001), MRSA252 (Holden et al., 2004), UAMS-1 (Cassat, 2006), JH1, and JH9 (Sieradzki et al., 2003). These strains represent the historical evolution of HA-MRSA as well as its geographic diversity, and encompass two main pulse field gel electrophoresis (PFGE) subtypes, USA100 and USA200 (Thurlow et al., 2012). HA-MRSA traditionally causes surgical site infections (SSIs), osteomyelitis, and septicemia in hospital patients.

CA-MRSA, on the other hand, was first isolated in the late 1990's when healthy individuals within the community, who had had no recent contact with a hospital setting, contracted fatal S. aureus infections (CA-MRSA) (Thurlow et al., 2012). It was later determined that these isolates were distinct from their HA-MRSA counterparts in that they mostly cause skin and soft tissue infections (SSTI's), occasionally cause necrotizing pneumonia, and can readily infect healthy individuals (Millar et al., 2007). The most commonly utilized CA-MRSA strains studied in the laboratory are SF8300 (Charlebois et al., 2002), LAC (Kennedy et al., 2008), FPR3757 (Diep et al., 2006), TCH1516 (Gonzalez, 2005), and MW2 (Baba et al., 2002). SF8300, LAC, FPR3757, and TCH1516 all belong to the dominant CA-MRSA PFGE subtype, USA300, while MW2 belongs to the second most common CA-MRSA PFGE subtype, USA400 (Thurlow et al., 2012). Additional CA-MRSA lineages are new recognized in the literature, but laboratory work characterizing these strains has only recently been undertaken (e.g. ST239).

While the variability in pathogenesis between HA and CA-MRSA isolates implies detectable genetic differences between these two MRSA sub-groups, CA-MRSA isolates exhibit a large amount of heterogeneity world-wide, making it difficult to suggest any single molecular test for identification of CA-MRSA isolates. Furthermore, CA-MRSA strains have begun to replace HA-MRSA strains in many hospitals around the world, confounding the traditional classification of MRSA isolates into the HA and CA categories (Popovich et al., 2008; Jenkins et al., 2009; Moore et al., 2009; Hultén et al., 2010). Thus, MRSA isolates have more recently been categorized into HA and CA sub-categories based on sequence typing (i.e. phylogeny) and pulse-field gel electrophoresis, of digested genomic DNA (Enright et al., 2000; McDougal et al., 2003). However, some simple molecular characteristics do separate CA-MRSA isolates from their HA-MRSA counterparts, albeit these characteristics are not shared by every CA-MRSA strain (Table 2) (Thurlow et al., 2012; Li et al., 2012).

Basic Protocol 1: GROWTH OF S.AUREUS ON SOLID MEDIA

S. aureus forms small, shiny, gold colonies on most solid media after 1 day of growth at 37°C. The size and color of the colonies is media and strain dependent (media containing low levels of glycolytic carbon sources will produce colonies that are less pigmented as are some strains harboring specific mutations that affect pigmentation, Figure 1). Several different media have been described for growing S. aureus including: Brain Heart Infusion (BHI) Agar, Tryptic Soy Agar (TSA), Todd Hewitt Agar (THA), Luria-Bertani (LB) Agar, Mueller-Hinton Agar (MHA), and Blood Agar. BHI, TSA, THA, MHA, LB Agar are all rich media and will allow for robust growth of S. aureus. Blood agar is also a rich media but allows for the additional observation of hemolysis. Most strains of S. aureus are β-hemolytic (i.e. produce complete lysis of red blood cells resulting in a zone of clearing, Figure 2) as a result of alpha toxin production. S. aureus colonies grown on blood agar are small, shiny, pigmented (to varying degrees depending on the glucose content of the blood agar used and the strain of S. aureus), and surrounded by a zone of clearing. Antibiotics can be added to the media as necessary according to the concentrations outlined in Table 3. It should be noted that USA300 isolates contain plasmids carrying an erythromycin and tetracycline resistance cassettes and therefore genetic manipulation of USA300 involving antibiotic selection is restricted.

Figure 2.

Figure 2

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Staphylococcus aureus strain SF8300 grown on Tryptic Soy Agar with Sheep's Blood for 18 hr at 37°C.

Materials

  • S.aureus frozen stock (Basic Protocol 3)

  • BHI Agar plates

  • Sterile wooden applicators

  • 37°C incubator

  1. Using a sterile wooden applicator streak out a small amount of S. aureus from the frozen stock onto a small section of a BHI plate (~1/4) using aseptic technique.
    Only a small amount of frozen stock is needed, enough to be visible at the end of the stick.

  2. With a new sterile wooden applicator, streak out a new quadrant of S. aureus by passing through the initial quadrant several times. Repeat this process 1–2 more times, passing a new applicator through the most recently streaked quadrant.

  3. Incubate the plates for 16–24 hrs at 37°C.

Basic Protocol 2: TESTING S. AUREUS FOR METHICILLIN RESISTANCE

As stated in Strategic Planning, S. aureus resistance to penicillinase-stable penicillins (i.e. methicillin, oxacillin, nafcilin, etc.) is encoded by the mecA gene located on the SCCmec cassette (Ubukata et al., 1989). The most accurate method for identifying methicillin-resistant Staphylococcus aureus (MRSA) is to test for the presence of the mecA gene by PCR (Unal et al., 1992). However, phenotypic confirmation of methicillin resistance is highly recommended regardless of the outcome of the PCR reaction as strains carrying the mecA gene can exhibit susceptibility to methicillin (Araj et al., 1999; Bignardi et al., 1996). The primary methods for demonstrating S. aureus methicillin resistance are the cefoxitin disk screen test, the latex agglutination test for PBP2a (the enzyme encoded by mecA), and the Oxacillin-salt agar screen (Sakoulas et al., 2001). While the latex agglutination test for PBP2a has been shown to be the most accurate method for determining S. aureus resistance to methicillin, the Oxacillin-salt agar screen is the simplest method and is still very accurate (99% sensitivity and 98.1% specificity) (Sakoulas et al., 2001). According to the Clinical and Laboratory Standards Institute (CLSI) S. aureus strains exhibiting growth on MHA supplemented with 2% NaCl and ≥ 4 μg/ml Oxacillin are considered methicillin-resistant while S. aureus strains only exhibiting growth on MHA supplemented with 2% NaCl when Oxacillin-levels are ≤ 2 μg/ml are considered methicillin-susceptible. Additional information regarding the assay described below can be found in the Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement published by the CLSI (document M100-S17).

Materials

  • S.aureus streak plate (Basic Protocol 1)

  • Mueller-Hinton Agar (MHA) plate supplemented with 2% NaCl and Oxacillin (2 and 4μg/ml)

  • 0.5 McFarland Standard

  • Sterile Microfuge tubes

  • Plastic Cuvettes

  • Vortexer

  • Spectrophotometer

  • 35°C incubator

  1. Using aseptic technique, transfer several well isolated colonies of S. aureus from the streak plate (plate prepared in Basic Protocol 1) into 1ml of PBS, contained in a 1.5 ml microfuge tube, by rubbing the inoculating loop against the side of the tube at the liquid-air interface.
    There should be enough bacteria to be visible on the loop.

  2. Thoroughly resuspend the transferred S. aureus by vortexing the mixture or pipetting up and down repeatedly.

  3. Adjust the turbidity of the S. aureus suspension to that of a 0.5 McFarland Standard using a spectrophotometer.
    McFarland Standards are a turbidity reference for the dilution of bacterial suspension into a range of desired concentrations. McFarland Standards can be prepared by mixing barium chloride with sulfuric acid (McFarland Standard 0.5: 0.05ml 1% barium chloride with 9.95ml 1% sulfuric acid) or from purchased suspensions of latex particles (greater accuracy and longer shelf life). The S. aureus suspension should be diluted with sterile PBS to achieve an absorbance equal to the 0.5 McFarland Standard using a wavelength of 600 or 625nm.

  4. Aliquot 10μl of the diluted S. aureus onto the surface of a MHA-salt plate, a MHA-salt plate with 2μg/ml Oxacillin, a MHA-salt plate with 4μg/ml Oxacillin.

  5. Allow spots to dry and then incubate the plates at 35°C for 24 hrs.

  6. Methicillin-resistant Staphylococcus aureus (MRSA) will exhibit growth on all three plates while methicillin-susceptible Staphylococcus aureus (MSSA) will grow on all plates except the MHA-salt plate with 4 μg/ml Oxacillin.

Basic Protocol 3: GROWTH OF S. AUREUS IN LIQUID MEDIA

S. aureus grows rapidly in broth culture at 37°C with aeration. As such, overnight cultures of S. aureus are typically started at the end of the day just prior to leaving the lab using either an isolated colony from an agar plate (Basic Protocol 1), a second broth culture, or a frozen stock (Basic Protocol 4). Several different media have been described for growing S. aureus in broth culture, including: Brain Heart Infusion (BHI), Tryptic Soy Broth (TSB), Todd Hewitt Broth (THB), Luria-Bertani (LB) Broth, and Chemically Defined Media (CDM). There is no specific advantage or disadvantage to using BHI, TSB, THB, or LB; all are rich media and will allow for S. aureus to grow to a high concentration overnight. CDM, on the other hand, is generally utilized for growth experiments involving the limitation or addition of specific nutrients. Regardless of the media, it should be noted that S. aureus is auxotrophic for Arginine (Emmett and Kloos, 1979) and Proline (Li et al., 2010), that S. aureus cannot use inorganic sulfur sources (e.g. sulfate or sulfite) (Soutourina et al., 2009), and that S. aureus exhibits a preference for glycolytic carbon sources during aerobic growth (Seidl et al., 2009). Repeated passaging of S. aureus in liquid media can result in the accumulation of mutations and thus, should be avoided. Overnight cultures of S. aureus will exhibit gold pigmentation, although the extent of pigmentation is media and strain dependent (Figure 3).

Materials

  • BHI broth

  • Streak plate of S. aureus

  • Wire inoculating loop

  • Sterile 15–20ml culture tubes (with caps)

  • 37°C Incubated shaker

  1. Using aseptic technique, transfer a single colony of S. aureus from the streak plate (plate prepared in Basic Protocol 1) into the aliquoted broth by tilting the culture tube and rubbing the inoculating loop against the side of the tube at the liquid-air interface.
    There should be enough bacteria to be visible on the loop.
  2. Grow cultures overnight (~16–18hrs) at 37°C with shaking (250rpm).
    S. aureus is a facultative anaerobe. Thus, the overnight culture does not have to be shaken at 250 rpm, however the growth rate and maximum bacterial density achieved may be affected. To maximize aeration, place the tube on the shaker at an angle. Following overnight growth, the culture will be in stationary phase.

Basic Protocol 4: PREPARATION OF S. AUREUS FROZEN STOCKS

Long-term storage of S. aureus should occur at −80°C to prevent accumulation of mutations. To prepare a frozen stock of S. aureus add an aliquot of an overnight culture (Basic Protocol 3) to sterile DMSO as described below. The addition of DMSO prevents complete freezing of the cells and thus limits cellular damage as a result of the transition to −80°C. Sterile glycerol (final concentration 25–50% once mixed with bacteria) is frequently used as an alternative to DMSO. There is no distinct advantage to using one agent over the other except that DMSO thaws more slowly than glycerol, and thus maintains a low culture temperature for a longer period of time if freezer failure should occur.

Materials

  • Overnight culture of S. aureus (see Basic Protocol 3)

  • Sterile DMSO

  • Sterile 2ml cryotubes

  • −80°C Freezer

  1. Add 150μl of sterile DMSO to one sterile cryotube using aseptic technique.

  2. Add 850μl of overnight culture to the same cryotube using sterile technique.

  3. Vortex or invert briefly to mix.

  4. Store cryotube at −80°C.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Brain Heart Infusion (BHI) Agar Plates

Add 52g of BD BBL™ Brain Heart Infusion Agar to distilled water in a 2L flask. Alternatively, 40g of BBL™ Tryptic Soy Agar or BBL™ Luria-Bertani Agar can be similarly utilized. Add a stir bar to the flask and then place the flask on a stir plate to mix. Bring volume up to 1L using distilled water. Loosely cover the top of the flask with aluminum foil and then autoclave the media on liquid cycle (use 30 minute cycle to prevent glucose carmelization). Once sterilized, place the flask on a stir plate and stir the media at a low setting for ~45 minutes or until the media has cooled to 65°C. Add antibiotics as necessary and then pour the agar into sterile petri plates (~20–25ml/plate). Allow agar to solidify at room temperature for at least 1 day (minimizes condensation) then store the plates, top down, in plastic bags at 4°C.

Brain Heart Infusion (BHI) Broth

Add 37g of BD BBL™ Brain Heart Infusion to distilled water in a 2L flask. Alternatively, 30g of BBL™ Tryptic Soy Agar, 25g of BBL™ Luria-Bertani Agar or 30 g of BBL™ Todd-Hewitt Broth can be similarly utilized. Add a stir bar to the bottle and place the bottle on a stir plate to mix. Bring volume up to 1L using distilled water. Once the powder is completely dissolved, remove the stir bar, loosely cap the bottle, and autoclave the media on liquid cycle (do not tightly screw on the bottle cap or else the bottle will become highly pressurized in the autoclave; use 30 minute cycle to prevent glucose carmelization). Once sterilized, the broth may be stored at room temperature or 4°C.

Mueller-Hinton Agar Plates Supplemented with 2% NaCl and Oxacillin (MHA-salt plates)

Add 38g of BD BBL™ Mueller-Hinton II Agar and 20g NaCl to distilled water in a 1L bottle. Add a stir bar to the flask and then place the flask on a stir plate to mix. Bring volume up to 1L using distilled water. Loosely cover the top of the flask with aluminum foil and then autoclave the media on liquid cycle (use 30 minute cycle). Once sterilized, place the flask on a stir plate and stir the media at a low setting for ~45 minutes or until the media has cooled to 65°C. Add antibiotics as necessary (i.e. 2μg/ml and 4μg/ml Oxacillin) and then pour the agar into sterile petri plates (~20–25ml/plate). Allow agar to solidify at room temperature for at least 1 day (minimizes condensation) then store the plates, top down, in plastic bags at 4°C.

Chemically Defined Media (CDM)

Make the solutions outlined in Table 4 and sterilize as indicated (the amino acids and bases are made and stored separately to maximize the longevity of storage). Once cooled, all solutions should be stored at 4°C. Place a stir bar into a 250ml beaker and place the beaker onto a stir plate. Add 10mls of the salt solution, 1ml of each amino acid, 1ml of each base, 0.1ml of the vitamin solution, and 0.1ml of the trace elements solution to the beaker while slowly stirring the mixture. Add carbon sources as desired and then adjust the pH of the media to 7.4 using 10M NaOH. Bring the final volume of the media up to 100ml using distilled water and then filter sterilize the media.

STERILE DMSO

Sterilize 100% DMSO using a syringe filter (0.2μm Nylon syringe filter attached to a 10ml syringe). Do not attempt to sterilize the DMSO using a nitrocellulose filter because it will dissolve once it is exposed to the solvent.

COMMENTARY

Background Information

S. aureus was first isolated from a knee abscess in 1881 (Ogston, 1881). Since then, S. aureus has become the most dominant nosocomial infection in the developed world (i.e. the MRSA epidemic). Although the prevalence of HA-MRSA amongst hospital-acquired infections is currently waning, new subsets of MRSA (USA300, USA400, ST239, etc.) have arisen within the community setting (Climo, 2009). These community-acquired MRSA (CA-MRSA) isolates exhibit increased capacity to cause disease in healthy individuals (Weber, 2005). The primary clinical presentation of USA300 is skin and soft-tissue infections, but it has also been found to cause fatal necrotizing pneumonia (Millar et al., 2007).

MRSA as a whole is capable of causing a wide variety of clinical presentations, including: pneumonia, surgical site infections, endocarditis, myocarditis, osteomyelitis, toxic shock syndrome, scalded skin syndrome, and septicemia. The ability of S. aureus to cause such varied diseases is largely attributed to the diversity of virulence factors encoded in the S. aureus genome and the innate resistance of S. aureus to nearly all facets of the human immune system. One such virulence factor produced by S. aureus is the distinct yellowish-orange pigment called staphyloxanthin (aureus means “golden” in Latin). Pigment production has been shown to aid in the resistance of S. aureus to oxidative damage (Katzif et al., 2005; Pelz et al., 2005; Liu, 2005). Other major virulence factors encoded by S. aureus include: toxins (e.g.hla, hld, hlg, set8, psmα), proteases (e.g. spl, sspC, aur), lipases (lip and geh), adhesins (e.g. cflA, cflB, fnbA, fnbB), coagulases (coa and vwb), and factors involved in immune resistance (e.g. ldh1, hmp, katA, spa, eap, chp, sak, and capsule) (Foster, 2005; Foster and Höök, 1998; Dinges et al., 2000).

Aside from these obvious virulence factors, S. aureus is also a fast growing organism that is capable of utilizing a variety of carbon sources (interestingly, USA300 isolates grow faster in vitro than their HAMRSA counterparts, (Thurlow et al., 2012)). As mentioned earlier, all S. aureus strains exhibit an in vitro auxotrophy for arginine and proline, although for proline, this is only during growth on glycolytic carbon sources (e.g. glucose). This is caused by catabolite-mediated repression of the proline biosynthetic genes via the catabolite response regulator, CcpA (Li et al., 2010). Thus, careful consideration of the growth media chosen for experimentation should be taken, given that S. aureus exhibits extensive metabolic regulation.

Critical Parameters and Troubleshooting

Proper growth and maintenance of S. aureus in the laboratory is not difficult. Inocula need not be large in order to generate robust growth, and chances for contamination are rare given the short incubation times/fast growth rate of S. aureus cultures. Furthermore, contaminants are easily discernible from S. aureus by several easily testable phenotypes, all of which S. aureus is generally positive for, including: gold pigmentation, catalase activity, coagulase activity, β-hemolysis, and growth on Mannitol salt agar. Repeated passage of S. aureus in the laboratory is not recommended as it may result in the accumulation of undesired mutations.

Anticipated Results

For growth on BHI agar, S. aureus should appear as small, shiny, gold colonies after 12–24 hrs of growth at 37°C. Likewise, overnight cultures of S. aureus in BHI broth should appear golden and highly turbid.

Time Considerations

S. aureus is a fast growing microorganism. Liquid cultures generally need only 12–24hrs to achieve a high density while most strains of S. aureus will become visible as colonies on a plate after 12–36hrs of growth at 37°C.

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